An experimental method is generally known wherein a desired location of a sample is irradiated with a laser beam (referred to herein as the ‘excitation beam’) for excitation of the sample while observing/recording reflected observation light and fluorescence from the sample so that changes in the appearance of the sample may be observed and/or recorded.
A laser scanning microscope for this purpose is disclosed in Japanese Laid-Open Patent Application H10-206742, which also discloses a scanning method. According to this scanning method, the entire sample may be scanned with an observation beam emitted by a first laser light source using a first scanning optical system while an arbitrary location of the sample is irradiated with an excitation beam emitted by a second laser light source using a second scanning optical system, and fluorescence from the sample may be detected with a photoelectric conversion element. In addition, other scanning methods may be used with a confocal laser scanning microscope, such as a method to deflect light utilizing a photo acoustic element, a method to deflect light using a galvanometer mirror, a method to direct light by rotating a pinhole disk called a Nipkow disc, and so on.
An example of the construction of a conventional laser scanning microscope that uses two pairs of galvanometer mirrors for scanning will now be discussed. As shown in FIG. 6, in a laser scanning microscope 10, a first scanning optical system 1 for observation includes an observation beam source 11 that emits an observation beam 22, a collimator lens 12, and a pair of galvanometer mirrors 14a, 14a′ that perform (in the x-axis and y-axis directions) a two-dimensional scan of the observation beam 22. On the other hand, a second scanning optical system for excitation 2 includes an excitation beam source 19 that emits an excitation beam 23, a collimator lens 12, and a pair of galvanometer mirrors 14b, 14b′ that perform, in the x-direction and y-direction, a two-dimensional scan of the excitation beam. In addition, a dichroic mirror 13a is positioned at a location where the observation beam 22 and the excitation beam 23 intersect one another. The dichroic mirror 13a transmits the observation beam 22 and reflects the excitation beam 23 so as to combine the two beams on a common optical path. A relay lens 15, an image-forming lens 16, and an objective lens 17 are placed along the common optical path, and a sample 18 is placed at the focal point of the objective lens 17. Also a dichroic mirror 13b is placed between the collimator lens 12 and the galvanometer mirror 14a in the light path of the first scanning optical system 1 so as to direct light reflected/emitted from the sample 18 (i.e., after the sample has been irradiated with the observation beam 22 and the excitation beam 23) to a detection optical system 3. The detection optical system 3 is formed of an image-forming lens 16, a pinhole 20, and a photoelectric conversion element 21. The pinhole 20 is used to block light rays other than those rays reflected/emitted at a conjugate point on the sample which is illuminated by the observation beam, since the pinhole 20 is positioned along the optical axis of light from the sample that has been reflected by the dichroic mirror 13b, and the pinhole 20 is positioned at the focal plane of the image-forming lens 16. In this way, optical information from the sample 18 can be measured with the photoelectric conversion element 21 which, for example, may be a photo multiplier.
Because the pair of galvanometer mirrors 14a, 14a′ for the observation beam 22 and the pair of galvanometer mirrors 14b, 14b′ for the excitation beam 23 are located and controlled independently, scanning of the observation beam and the excitation beam may be executed simultaneously and independently. For example, FIG. 7 shows a conventional sample scanning method using the observation beam 22 and the excitation beam 23, wherein the two galvanometer mirrors 14a, 14a′ (see FIG. 6) of the first scanning optical system 1 are operated so as to scan the sample in two dimensions using the observation beam 22, and further, the two galvanometer mirrors 14b, 14b′ of the second scanning optical system for excitation 2 are simultaneously operated in order to scan the sample in two dimensions using the excitation beam 23.